Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that no nucleic acid is necessary to allow for the infectivity of a prion protein to proceed.
A major step in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bolton, McKinley et al. (1 982) Science 218:1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983) Cell 35:57-62]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP.sup.C is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrP.sup.C is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases result from the transformation of the normal form of prion protein (PrP.sup.C) into the abnormal form (PrP.sup.Sc). There is no detectable difference in the amino acid sequence of the two forms. However, PrP.sup.Sc when compared with PrP.sup.C has a conformation with higher .beta.-sheet and lower .alpha.-helix content [Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) J Biol Chem 268:20276-20284]. The presence of the abnormal PrP.sup.Sc form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrP.sup.Sc plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it is a critical factor in neuronal degeneration [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition 103-143]. The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172:21-38]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Sheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) N Engl J Med 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
Prions exist in multiple isolates (strains) with distinct biological characteristics when these different strains infect in genetically identical hosts [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 165-186]. The strains differ by incubation time, by topology of accumulation of PrP.sup.Sc protein, and in some cases also by distribution and characteristics of brain pathology [DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6th Edition:235-280]. Because PrP.sup.Sc is the major and very probably the only component of prions, the existence of prion strains has posed a conundrum as to how biological information can be enciphered in a molecule other than one comprised of nucleic acids. The partial proteolytic treatment of brain homogenates containing some prion isolates has been found to generate peptides with slightly different electrophoretic mobilities [Bessen and Marsh (1992) J Virol 66:2096-2101; Bessen and Marsh (1992) J Gen Virol 73:329-334; Telling, Parchi et al. (1996) Science 274:2079-2082]. These findings suggested different proteolytic cleavage sites due to the different conformation of PrP.sup.Sc molecules in different strains of prions. Alternatively, the observed differences could be explained by formation of different complexes with other molecules, forming distinct cleavage sites in PrP.sup.Sc in different strains [Marsh and Bessen (1994) Phil Trans R Soc Lond B 343:413-414]. Some researchers have proposed that different prion isolates may differ in the glycosylation patterns of prion protein [Collinge, Sidle et al. (1996) Nature 383:685-690; Hill, Zeidler et al. (1997) Lancet 349:99-100]. However, the reliability of both glycosylation and peptide mapping patterns in diagnostics of multiple prion strains is currently still debated [Collings, Hill et al. (1997) Nature 386:564; Somerville, Chong et al. (1997) Nature 386:564].
A number of methods exist for the detection of a protein in a sample, and specifically for the detection of PrP.sup.Sc. Assays to detect PrP.sup.Sc are described in U.S. Pat. Nos. 5,565,186 and 5,792,901 and U.S. patent application Ser. No. 08/935,363, incorporated herein by reference, which describe and disclose immunoassay methods for determining the presence of PrP.sup.Sc in a sample. Quality assurance, quality control, and reagent documentation are all critical issues in determining the presence of infectious prions in a sample. Variation between assays can be reduced by the use of a common standard for the calibration of the different methods. The basis of a calibration system is a primary standard sample that provides both high sensitivity and reproducibility of detection to effectively and consistently analyze different samples. A standard is indispensable in assigning an accurate target value to reference materials in an assay method. Standards are also useful in testing reagents used in assays for reliability and effectiveness.
There is a method of providing standardized, cost-effective assays for reproducibly testing sample materials for the presence of a prion protein. Accordingly, there is a need for standards for the calibration of assays to detect prions and as controls in the assays, to ensure high sensitivity and to reduce problems of irreproducibility between different samples, and to test the quality of reagents used in the assays.